The anion exchange protein (band 3) is an abundant protein in the human erythrocyte membrane which is composed of two domains that serve distinct functions. The integral membrane domain facilitates the exchange of bicarbonate and chloride, thereby increasing the CO2 carrying capacity of the blood. The cytoplasmic domain serves as a binding site for peripheral proteins including ankyrin and band 4.2 which are involved in forming a physical link between the plasma membrane and the membrane skeleton. These protein-protein interactions are essential for maintaining the unique biconcave shape and viscoelastic properties of erythrocytes. Genetic defects in band 3, ankyrin, or band 4.2 result in abnormal cell shapes, rigid or fragile cells, and often, clinically significant hemolytic anemias. The long- term goals of these studies are to determine the structural and dynamic properties of band 3 that are essential for its role as an organizing center for protein-protein interactions which stabilize the erythrocyte membrane. It is hypothesized that band 3 is present primarily as dimers and tetramers, that these small oligomers interact with the membrane skeleton via a flexible linkage, and that the flexibility of the cytoplasmic domain of band 3 is necessary for normal cell shape and mechanical properties. In the next grant period, electron paramagnetic resonance, fluorescence, and phosphorescence spectroscopies will be combined with classical biochemical methods to address the following specific questions: 1) What are the sizes of the oligomeric species of band 3 present in the erythrocyte membrane? 2) How flexible, in terms of the amplitude of motion, is the segment of the cytoplasmic domain of band 3 which links the membrane skeleton to the lipid bilayer, and to what extent do these links exist in the intact cell? 3) What are the properties of specific protein-protein interactions involving the cytoplasmic domain of band 3 and the binding proteins ankyrin and band 4.2? To help accomplish these goals, new molecular probes, spectroscopic methods, and data analysis methods will be developed which will be of utility in the biomedical research community.